The objective of this grant application is to find strategies to manipulate T-cell-mediated immunity by controlling the differentiation and function of Foxp3+ regulatory T cells (Treg). Foxp3 is a forkhead transcription factor, predominantly expressed in CD4+CD25+ Treg cells, and mediates not only differentiation but also functional roles in these cells. This Treg cell development was induced via Foxp3 expression by a retroviral system, indicating that we can control Treg cell differentiation by regulating Foxp3 gene expression. Treg cells play an important role in suppressing unwanted immune responses, including the prevention of autoimmune diseases caused by potentially hazardous self-reactive T cells. Some tumors, however, can also escape from the immune system due to the suppressive activity of Treg cells. Controlling the differentiation and function of Treg cells via the molecular mechanisms of Foxp3 expression might offer potential therapeutic targets for the modulation of immune responses in these diseases. The mechanisms underlying Foxp3 gene expression, however, have not been described until recently, because the study of this gene expression was limited by the small number of Treg cells in mononuclear cell populations. We generated a model system for analyzing Foxp3 expression in a T cell line and, using this system along with primary T cells, identified two enhancers in the mouse Foxp3 gene. One of the enhancers contains the Smad3 and NFAT binding sites and plays a key role in the induction of Foxp3 transcription. Our recent study also suggests that more transcription factors are involved in regulation of Foxp3 expression through complicated molecular mechanisms. Inhibition of Foxp3 expression by IL-6 and OX40L has been previously reported, and very recently we found that GITRL also strongly inhibits it. Furthermore, we found that ICN (active form of Notch) upregulates the Foxp3 promoter activity but inhibits one of the enhancer activities. One regulatory element identified at the 5'-UTR may play a key role for "Cross-talk" between the promoter and enhancers. Foxp3 gene expression seems to be regulated by many transcription factors and their interactions. The proposed application offers three specific aims: (1) To identify transcription factors involved in Foxp3 gene expression, (2) To determine interaction of transcription factors in the Foxp3 promoter and enhancers, and (3) To determine contribution of transcription factors binding to Foxp3 promoter and enhancers in vivo. Public Health Relevance: Treg cells play an important role to prevent autoimmune diseases. However, some tumors seem to escape from the immune system using the suppressive activity of Treg cells. Treg cell development is regulated by Foxp3 expression, indicating that controlling the differentiation and function of Treg cells via the molecular mechanisms of Foxp3 expression might offer potential therapeutic targets for the modulation of immune responses in these diseases.